Lec. 6.   Biol C2005/F2401   2000   L. Chasin     September 21, 2000

(C) Copyright 2000   Lawrence Chasin and Deborah Mowshowitz   Department of Biological Sciences   Columbia University   New York, NY
Last recorded update:  Tuesday, September 26, 2000 09:15 PM

Exam #1 is Thursday evening, Sept. 28, 1999, at 7:00 or  5:40, depending on your class and/or last name.  Click here for your room/time assignment.

There will be a review session (with Dr. Chasin) to answer your questions on Thursday 9:10 - 10:25 AM in 309 Havemeyer.

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Protein purification methods:
    ultracentrifugation
    native gel electrophoresis
    SDS-gel electrophoresis
    molecular sieve chromatography

Membrane proteins

Proteins bind molecules with great specificity

Protein domains

Catalysis

Activation energy

Substrates & products

Chemical kinetics

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(Protein purification methods)
PROTEIN PURIFICATION/SEPARATION METHODS

(ultracentrifugation)
  Here is one sometimes useful method: ULTRACENTRIFUGATION ULTRACENTRIFUGATION

Ultra means = >20,000 rpm; 60,000 rpm is common, compare a Ferrari revving at 6000 rpm, red-lining; this is ten times faster; you need a vacuum chamber so no heat from air friction will be produced.

A mixture of molecules will be subject to two main forces in the ultracentifuge as it starts to spin (ignoring buoyant force):
Causing sedimentation is the centrifugal force = m(omega)²r = (which is proportional to the mass or MW of a protein). 
    m = mass, omega = angular velocity, and r = distance from the center of rotation.
Opposing sedimentation = friction = f_oV.
    f_o = frictional coefficient, a constant for any particular protein, it is minimum for a sphere, higher for less compact shapes like cigars or pancakes.
    V = velocity of the molecule as it moves away from the center of rotation .

Soon after accelerating, V increases to a point where no further acceleration takes place, as the forces on the molecule are balanced. It continues to sediment, but at a constant velocity.

Now at this point, at this velocity: Centrifugal Force = Frictional force (there's no net force, no acceleration, but constant velocity)

So at this point (soon achieved):  M(omega)²r =  f_oV

And:  V = m(omega)²r/f_o,

where f = a frictional coefficient dependent on shape
(to visualize the effect of shape on friction, compare the velocity of a falling feather vs. a tiny pebble of equal weight, dropped in the fluid of air).

Higher f = more friction.

If assume a spherical shape, then we can estimate a MW (Assume  f_o,  and then measure V and r, so we can solve for m, or the MW)

On the other hand, if we know the MW, we can get information about shape (via f_o).

Sedimentation velocity is often measured in Svedbergs, which takes the centrifugation conditions into account s = V/(omega)²r, and so m = sf_o.

So ultracentrifugation separates proteins on the basis of MW and shape.  It is a gentle procedure (non-denaturing, can be carried out at nice low temperature (say 4 deg C, which tends to stabilize proteins) and in the presence of a buffer at pH 7 and physiological levels of salts).

You can recover your protein by punching a hole in the bottom of the centrifuge tube, and collecting the solution in a series of tubes as it drips out the bottom. Each tube can then be examined, or assayed, for the presence of the protein to be purified. For this purpose you need to be able to detect the protein in the midst of the other proteins. For example, if you were purifying Anfinsen's ribonuclease, you could measure the ability of the tube contents to catalyze the breakdown of RNA  to its monomers.

How about separation on the basis of the net charge of a protein. We separated amino acids on the basis of charge in paper electrophoresis. For proteins, the solid supporting material is a gel, not paper:

GEL ELECTROPHORESIS:

There are two types -

( native gel electrophoresis)
First: native gel electrophoresis

acrylamide, a reactive chemical (a monomer in this chemistry) in aqueous solution can be made to polymerize into  polyacrylamide (thus polyacrylamide gel electrophoresis, or P.A.G.E.). The result is a network of polymer fibers, which form a gel, with about the consistency of Jello.

Usually carried out in an apparatus in which this gel stands vertically supported in a sandwich between two glass plates. The top of the gel is connected by a salt solution to an electrode, as is the bottom edge of the gel. A protein mixture is applied to the top of the gel slab.

The electrophoresis is started by applying a voltage (~200 v) across the gel.

The rate of migration of a protein in the gel depends on two properties:
The first is its net charge.
Molecules with the most charge (of a sign opposite to that of the far electrode) will migrate to the far electrode fastest.  

Second, its "size" (which is proportional to MW if spherical). The gel consists of a network of fibers. Depending on the concentration of polyacrylamide, the network canbe dense or tight, so that the proteins have trouble migrating, as they must negotiate their way through the tangled fibers. Molecules that are smallest (i.e., lowest MW) will worm their way through the gel fibers fastest. 
So the smallest and most highly charged protein wins the race. 
After the electrophoresis has been stopped, molecules will be distributed along the gel length according to these two characteristics (MW and net charge).  For instance, a highly charged protein molecule, although pulled with  a greater electromotive force, will not have gotten very far if it is relatively large. 
[Note that molecules with a charge opposite to the near electrode, will migrate up and off the gel, into the buffer reservoir and be lost.  Trial and error will dictate how you setup the electrophoresis if you do not know the charge on the protein you are trying to isolate.] 

Sometimes the gel is made purposely loose, so that the effective pore size is very large and then charge alone determines mobility. Starch gels rather than polyacrylamide have been used to create large pore gels. 

(SDS gel electrophoresis)
A second, a more widely used variation of gel electrophoresis is SDS PAGE.

Here sodium dodecyl sulfate, or SDS (or SLS) is included in the gel: CH₃-(CH₂)₁₁- SO₄⁼

[sulfate is similar in structure to phosphate, and is a strong acid].  Like a phospholipid, SDS has a highly polar end and a highly hydrophobic body.

Might you expect SDS to denature a protein? Yes. It's a detergent and a powerful denaturant. It binds all over the protein, coating every protein with a nearly uniform negative charge.  SDS is put into in the gel when you form it and into the electrophoresis buffer. Now when SDS-PAGE is run, where should the anode  be placed? Does it matter? Yes, the protein is coated with negative charge now, so the anode is always the far electrode, put at the bottom.

Under these denaturing conditions, the polypeptides exist as a random coils, which then migrate solely on the basis of their size, which is the equivalent of a sphere for all polypeptides. Larger molecules have more difficulty finding their way through the polyacrylamide fibers.  So the lowest MW wins always wins in SDS-PAGE.

If you run standards of known MW, you can determine the MW of your protein by comparison, and this is a very common way to assign a MW to a polypeptide. However, it is not always completely accurate, as some proteins probably do bind a bit more SDS than others. And one must remember to reduce the disulfides with mercaptoethanol first (usually), so as to have a truly random coil for comparisons with other proteins.

If you don't yet know what a protein does, you can just call it by its molecular weight, from SDS gels: e.g., p53, a famous protein whose absence is associated with cancer was named this way, and the name has stuck even though quite a lot is known about its function now (p in p53 stands for protein, so you have names like p27, p100 etc.).

(Gel filtration)
If you have a protein with quaternary structure, SDS-PAGE will give you the MW of the polypeptide subunits, since the SDS will denature the protein and so dissociate it into its subunits  If you want to know the MW of a protein in its native quaternary structure, you need a method seprates proteins under mild conditions that maintain its native structure. 

For this we could use molecular sieve chromatography, also called or Sephadex chromatography , or gel filtration (these are all ~synonymous).

You start with plastic-like beads in a glass column with a support screen on the bottom.

You add your protein mixture to the top of the column material, then begin elution by adding a large volume of a buffer. The beads have been manufactured to be riddled with channels of a specified fairly uniform size. If a protein is smaller than the channel size, it enters a channel, explores it by diffusion, and eventually makes its way back out, having wasted its time in the race to the bottom of the column.  Larger proteins can't fit in to the channels, so they don't waste their time, and they  win the race. Intermediate sized proteins have a bit of trouble getting into a channel, so they waste some time but less than the smaller proteins. So larger molecules come out (elute) first, and the smallest come out last.  Here again, you would collect the eluted proteins in a series of tubes, and then assay each tube for the presence of the protein being purified.  If you calibrate the column by noting the behavior of spherical proteins of known size, you can determine the MW of your protein by comparison, if it is also spherical. If is is not spherical it will appear to have a higher molecular weight than its true MW (imagine a pancake being excluded from a channel while a sphere of the same MW gets in). 

Other methods include ion exchange chromatography, which also takes advantage if the net charge on a protein, and  affinity chromatography, which takes advantage of the surface properties of a protein (which we'll discuss next). One can purify a particular protein away from all other proteins in 4-5 such steps. For more on these protein separation techniques, see the protein separation handout.

Here are some images of laboratory ultracentrifuge , slab gel electrophoresis, gel filtration apparatuses.

One has to able to follow the protein of interest, to detect its presence in the presence of all others. Often use its functional properties for this, which brings us back to structure and function of proteins.

(Membrane proteins)
As an introduction into an example of the function of proteins, let's consider first a special class of proteins that do not follow some of the rules we have have seen that govern protein structure in general.  These are the MEMBRANE PROTEINS, the proteins that reside, not in the aqueous environment of the cytoplasm or the nucleus, but rather in the hydrophobic environment of the membranes of the cell, including the cell membrane.

In these proteins, the hydrophobic side chains are on the outside, as there are no hydrophobic forces present i to force then to coalesce. These  proteins can usually diffuse laterally in the lipid bilayer of the membrane [Purves 5.4], they can aggregate with each other with specificity, and they can become anchored via attachment to structural cytoplasmic fibers.  They can be nearly completely enveloped by the lipid bilayer, or they could be partially immersed, with a more conventional half (i.e., hydrophobics on the inside, hydrophilics on the outside) sticking out into the cytoplasm or on the outside of the cell.  
See [Purves 5.1], [Purves 5.5].

One class of membrane proteins act as channels through the membrane.  The channel proteins are formed into cylinders, with a hydrophobic exterior, but with hydrophilic groups lining the hole through the interior of the cylinder.
Small molecules can pass through the cylinder, or channel, but large molecules cannot fit through (note: macromolecules CANNOT diffuse onto cells).

So we have a cellular function for a protein, and a function that shows some selectivity on the basis of size there. How about other criteria of selectivity, like charge?

Some channels can distinguish charge: + repels +, and attracts -, so a channel that is lined with positive charge could bind a  negatively charged ion and, if there's a higher concentration outside than inside, eventually pass these ions along from the outside of the cell to the inside. A positively charged ion on the other hand would be repelled by the channel and so would not get into the cell by this route.

You can a imagine the same sort of selectivity based on hydrogen bonding, for example.